In order to generate mouse models of human MYH9-related disease, which is caused by mutations in nonmuscle myosin IIA, and to study the pathological mechanisms of the mutations in these diseases, we generated 3 mouse lines, each with a different mutation in the nonmuscle myosin II-A gene, Myh9 (R702C, D1424N, and E1841K). Each line develops MYH9-related disease similar to that found in human patients. R702C mutant human cDNA fused with GFP was introduced into the first coding exon of Myh9, and D1424N and E1841K mutations were introduced directly into the corresponding exons. Homozygous R702C mice die at embryonic day 10.5-11.5, whereas homozygous D1424N and E1841K mice are viable. All heterozygous and homozygous mutant mice show macrothrombocytopenia with prolonged bleeding times, a defect in clot retraction, and increased extramedullary megakaryocytes. Studies of cultured megakaryocytes and live-cell imaging of megakaryocytes in the bone marrow show that heterozygous R702C megakaryocytes form fewer and shorter proplatelets with less branching and larger buds. The results indicate that disrupted proplatelet formation contributes to the macrothrombocytopenia in mice and most probably in humans. We also observed premature cataract formation, kidney abnormalities, including albuminuria, focal segmental glomerulosclerosis and progressive kidney disease, and mild hearing loss. Our results show that heterozygous mice with mutations in the myosin motor or filament-forming domains manifest similar hematologic, eye, and kidney phenotypes to humans with MYH9-related diseases. In addition to using these mutant mice to study the relation between the nonmuscle myosin IIA mutation and disease, we plan to use various cells lines derived from these mice to study the effects of the mutation on basic properties of the cell. These include cytokinesis, cell-cell and cell matrix adhesion, cell polarity and cell migration. To gain clear insights into the distribution and function of different isoforms of nonmuscle myosin II (NMII) in normal mice, the enhanced GFP or mCherry sequence has been inserted in front of the start codon of the Myh9 gene in the first coding exon. We have obtained homozygous GFP or mCherry tagged NMIIA mice. The expression level of the tagged NMIIA is similar to that of the endogenously expressed untagged NMIIA in wild type mice. This fluorescence tagged NMIIA mouse model will shed light on the functions of NM IIA in development and in different cell types, tissues, and organs. Various cell lines derived from the mice will be used to study the regulation and function of NM IIA in adhesion, cell polarity and cell migration. For example, we have isolated bone marrow stem cells from GFP-NMIIA mice and sent them to Dr. Ana-Maria Lennon-Dumnils lab in Institut Curie, France to study the function of NM IIA in DC cell migration during the immune response. The purpose of the substitution experiment is to learn whether one isoform of NM II, specifically NM IIC1, can functionally replace a second one, NM IIA, in mice. Alternative splicing of pre-mRNA of NMIIC generates several isoforms. An alternative exon encoding 8 amino acids can be incorporated into loop 1 at amino acid 227 to form NM IIC1. Another alternative exon encoding 41 amino acids can be incorporated into loop 2 at amino acid 636 to form NM IIC2. In a few cases, both inserts can be incorporated to form NM IIC1C2. NM IIC1 is found in a variety of tissues such as liver, kidney, testes, brain, and lung. In vivo study also found that an enzymatically active fragment, HMM of NM IIC1 has increased actin-activated MgATPase activity and in vitro motility compared with HMM of NM IIC0, which has no insert . Among the four isoforms of NM IIC, NM IIC1s actin-activated MgATPase activity and in vitro motility are closer to NM IIA than other isoforms of NM IIC. Therefore, we chose NM IIC1 to replace Nm IIA in vivo. To replace NM IIA with NM IIC1 in the mouse model, homologous recombination was used to inactivate NM IIA by inserting the cDNA for NM IIC1-GFP into the first coding exon of the Myh9 gene. We have obtained heterozygous NM IIC replacing NM IIA mice. However, breeding of heterozygous mutant mice does not produce homozygous NM IIC replacing NM IIA mice. Homozygous embryos die around embryonic day 10.5. At both E9.5 and E10.5, AC/AC embryos are dramatically smaller than wild-type littermates and are developmentally delayed. Apoptotic cells were found in AC/AC embryos at E9.5 and E10.5, indicating the cause of embryonic death is apoptosis related. Embryonic explants from E9.5 embryos were cultured and the mouse embryo fibroblast (MEF) migration from the explants was recorded with time lapse microscopy. Compared with wild type explants, homozygous AC/AC MEFs have a much higher outgrowth speed. Images of MEF cultures show more of the AC/AC MEFs seem to be polarized than the wild type, having both more lamellipodia ruffling and long filopodia structures, indicating cells in migration. Immunostaining shows that myosin IIC can form filaments in AC/AC MEFs. Stress fibers in AC/AC MEFs were fewer and less organized. Focal adhesions in AC/AC MEFs are also fewer in number and smaller in size. These results indicate that NMHCII-C cannot replace II-As function during focal adhesion formation and maturation. Additional experiments such as transwell assays will be carried out to further study the kinetic properties in AC/AC MEFs.